Selected lines and inbred strains. Tools in the hunt for the genes involved in alcoholism.

In their quest to elucidate the genetic influences contributing to alcoholism, researchers have long used selected lines and inbred strains of rodents. Selected lines are obtained by repeatedly mating those animals within a population that show extremely high or low values of the desired trait. Inbred strains are generated by mating male and female siblings, irrespective of any particular trait, over several generations. Both of these approaches have provided researchers with extensive knowledge about the genetic and neurobiological mechanisms contributing to alcohol-related traits. However, the use of these models is associated with some limitations, mostly resulting from the inbreeding involved in generating such lines and strains. Nevertheless, these models can offer some advantages over other genetic approaches, such as the analysis of quantitative trait loci or the generation of transgenic and knockout mice.


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reeding techniques to generate animals with desired traits have long been a staple of genetic res e a rch, including alcohol-related re s e a rc h .Two of the oldest techniques for studying the genetics of alcohol-related traits in animals are analyses of selected lines and of inbred strains, usually of rats or mice.The use of these animal models long predates the present re volution in molecular biology, because it does not necessitate advanced biological techniques.Like newer models using molecular genetic techniques, howe ve r, these breeding approaches are based on the concept of manipulating an ani-m a l's genetic material and studying the resulting effects on a behavior of interest.
Alcohol studies using selected lines and inbred strains rely on the study of g roups, or populations, of animals that differ on a genetic level.At the same time, the animals' environment can be c o n t rolled rigorously in the laboratory setting.Under these conditions, comparisons of various populations allow alcohol re s e a rchers to investigate how genes can influence a wide variety of a l c o h o l -related behaviors (e.g., alcohol consumption, tolerance, and withdrawal) as well as physiological traits that may be important in mediating a l c o h o l's effects.This article briefly re v i ew s the strategies used in generating selected lines and inbred strains.The art i c l e then discusses some of the applications of these models as well as some of the limitations associated with their use.

Generation of Selected Lines
Se l e c t i ve breeding has long been used in a g r i c u l t u re to enhance d e s i red characteristics (i.e., phenotypes) in both plants and animals.In the laboratory, re s e a rc h e r s c reate selected lines by exploiting the natural variability inherent in an animal population and breeding those individuals that have either extre m e l y h i g h or extremely low values of the phenotype of interest.For example, in re g a rd to alcohol withdrawal, re s e a rchers might mate either animals exhibiting the most seve re withdrawal symptoms or animals exhibiting the mildest withdrawal sympt o m s .After several generations of selective breeding, the re s u l t i n g lines will demonstrate stable differe n c e s in the phenotype of interest.
The most widely studied alcoholrelated phenotype in selected lines has been free-choice alcohol drinking.Although many animals will not drink alcohol when given a choice b e t ween alcohol and w a t e r, great variability exists in the amount of alcohol individual animals within a population will consume.Se l e c t i ve b reeding for differences in this phenotype was first initiated in the late 1940s, when Ma rdones and colleagues d e ve l o p e d the UChA and UChB lines of rats that exhibit low and high levels of alcohol consumption, re s p e c t i vely (Ma rd o n e s and Se g ovia-Riquelme 1983).Si n c e then, re s e a rchers have generated numero u s sets of selected lines of rats and mice that differ in free-choice alcohol intake.
As with the molecular biological a p p roaches discussed in other art i c l e s in this journal issue, selected lines re s u l t f rom the experimental manipulation of genes that cause differences in a phenotype.When selection occurs tow a rd both high and low levels of the phenotype of interest (called bi-dire c t i o n a l selection), the two lines will show prog re s s i vely greater differences (i.e., dive rgence) throughout the generations with respect to the trait of interest and in the genes underlying this trait.With re s p e c t to other traits and their underlying genes, h owe ve r, the two lines should re m a i n s i m i l a r.Thus, in the case of fre e -c h o i c e alcohol consumption, selective bre e d i n g results in two lines that have differe n t variants (i.e., alleles) of the genes re l a t e d to drinking but similar alleles of genes u n related to drinking (e.g., coat color).1

Generation of Inbred Strains
In contrast with selected lines, in which u n related animals with similar characteristics are mated, or crossed, inbre d strains are created by crossing male and female siblings.The offspring of this c ross again are mated to each other and so on for 20 consecutive generations.The result of this pro c e d u re is a population of animals in which only one allele of eve ry gene is present (i.e., which is homozygous for eve ry allele).C o n s e q u e n t l y, all animals within an i n b red strain are genetically identical, akin to identical twins.Re s e a rchers can reliably study these populations, because their genetic makeup has been effect i vely fixed and will not change, exc e p t for changes resulting from spontaneous alterations (i.e., mutations) in an indiv i d u a l's genetic material.Any differe n c es that exist between individual members of a strain most likely can be attributed to environmental influences.
To study the role of the genes underlying the trait of intere s t, re s e a rc h e r s must assess and compare numero u s i n b red strains.Di f f e rences between diff e rent inbred strains having the same e n v i ronmental history can be attributed to genetic d i f f e rences.Alcohol re s e a rc h e r s h a ve used this approach since the late 1950s, when McClearn and Ro d g e r s (1959) published seminal articles demons t r a t i n g d i f f e rences in free-choice alcohol consumption among numerous inbre d strains of mice.In those studies, the l e vel of free-choice alcohol consumption d i f f e red over tenfold among the inbre d strains tested.Fu rt h e r m o re, because the animals we re raised under identical e n v ironmental conditions, these differences must have resulted from g e n e t i c d i f f e rences between the strains.Un l i k e selected lines, howe ve r, which should differ from each other only with re s p e c t to the traits related to the behavior under investigation, inbred strains will differ from each other on a wide va r i e t y of traits both related and unrelated to the behavior under investigation.Fo r example, whereas selected lines bred for high and low alcohol consumption should differ in the amount of alcohol ingested but not in an unrelated trait (e.g., coat color), inbred lines may differ in both alcohol consumption and the unrelated trait.

Applications for Selected Lines and Inbred Strains in Alcohol Research
Selected lines and inbred strains have p rovided two important contributions to studies exploring the genetics of a l c o h o l -related traits.First, these animal models have yielded extensive know ledge about the genetic underpinnings of individual differences in alcoholrelated traits.For example, these lines and strains have enabled re s e a rchers to formulate extensive theories on the causes of high alcohol intake in animals (e.g., Li et al. 1993).In principle, selected lines and inbred strains can be used to determine the genetic factors that are c o r related with differences in any alcoh o l -related behavior of interest as we l l as the effects of chance differe n c e s b e t ween the lines and strains (Cr a b b e et al. 1990;Falconer and Mackay 1996).
The systematic differences betwe e n animal strains or individual animals with respect to an alcohol-related trait (e.g., alcohol consumption level) frequently are caused by genes that affect multiple phenotypes (i.e., have pleiotro p i c e f f e c t s ).Such pleiotropic effects may hint at the causes underlying a cert a i n target trait.For example, both in selected lines and inbred strains of mice a highly consistent, negative genetic correlation exists between free-choice alcohol consumption and alcohol withdrawal.This means that selected lines and inbred strains which experience c o m p a r a t i vely mild alcohol withdrawal also drink more alcohol and vice ve r s a ( Metten et al. 1998).Such a corre l a t i o n indicates that the genes influencing the s e verity of alcohol withdrawal also affect alcohol consumption and implies that alcohol withdrawal discourages vo l u n t a ry alcohol drinking in mice.
Analyses of the genetic differe n c e s b e t ween selected lines exhibiting high and low alcohol consumption also have helped re s e a rchers assess neuro b i o l o g i c a l d i f f e rences between selected lines.Su c h studies have found consistent innate d i f f e rences between high-and lowconsuming selected lines (Mc Bride and Li 1998).For example, some animals f rom high-preferring lines that have n e ver been exposed to alcohol ( i .e ., a l c o h o l -n a i ve animals) show lower levels of the brain chemical (i.e., neurotransmitter) s e rotonin than do alcoholn a i ve animals f rom low -p referring lines.This type of analysis enables scientists to identify potential mechanisms underlying alcohol consumption and to distinguish those mechanisms fro m the effects of alcohol consumption on brain function.Scientists would have difficulty conducting these analyses in humans, because controlling for their alcohol-drinking history is impossible.When interpreting the results of such studies, howe ve r, one must always consider that free-choice alcohol consumption (or any alcohol-related phenotype) in ro d e n t s is a model for the human condition that likely reflects some but not all of the elements contributing to human alcohol use and alcoholism.
The second important contribution of inbred strains and selected lines to alcohol re s e a rch has been that these animal models can consistently exhibit a phenotype otherwise considered rare in the "o u t b red," or nonselected, animals c o m m o n l y used in laboratories.Fo r example, high free-choice alcohol consumption is an uncommon behavior in most rodents; accord i n g l y, studies on the effects of alcohol consumption would have to invo l ve many animals, most of which could not be used because they do not show the desired behavior.C e rtain inbred strains (e.g., C57/BL6 mice or Fa w n -Hooded rats), howe ve r, re q u i re little training to initiate alcohol consumption (George 1987), as do s e l e c t i vely bred rats or mice (Fro e h l i c h 1995; Grahame et al. 1999).A large p ro p o rtion of those animals will exhibit the trait of interest, allowing re s e a rc h e r s to perform experiments (e.g., assessing the environmental and physiological factors that affect alcohol intake or testing medications designed to re d u c e drinking) without having to eliminate many animals that do not meet the alcohol consumption criteria.

Potential Caveats of Animal Models
In addition to the previously mentioned fact that animal models likely prov i d e only an incomplete re p resentation of human behaviors as complex as alcohol use and abuse, several other potential p roblems exist in interpreting the findings of such re s e a rch.Crabbe and colleagues (1990) have examined extens i ve l y the caveats associated with experiments assessing genetic c o r relations in inbre d strains and selected lines.For example, re s e a rchers must consider several factors when trying to determine whether the c o r relation between two traits (e.g., a l c ohol consumption and alcohol withdrawal) actually arises fro m t h e p l e i o t ropic actions of the gene or genes that underlie both traits rather than fro m the actions of two unrelated genes.
The most important of these factors is that to the extent possible, inve s t i g a t o r s must ensure that differences among seve r a l selected lines or inbred strains are not caused by random differe n c e s re s u l t i n g f rom inbreeding.After repeated inbre e ding (which occurs in both selected lines and inbred strains), both alleles for many genes become fixed within a popula-tion-that is, all individuals in that population carry the same allele of a cert a i n gene.This fixation occurs both for genes that are re l e vant to the trait under inve stigation (e.g., alcohol consumption) and for genes that are irre l e vant to that trait.For example, among inbred mice, animals of the strain C57BL/6 (which re a d i l y drink alcohol) have a black coat, where a s animals of the strain DBA/2 (which a void alcohol) have a tan coat.T h u s , one could conclude that the same genes that determine coat color also d e t e r m i n e alcohol consumption levels.To support such a conclusion, h owe ve r, scientists must determine whether a consistent c o r relation between coat color and alcohol consumption exists in other inbre d strains as well.In fact, re s e a rchers typically must assess about 12 to 15 inbre d strains before the data have sufficient statistical power to detect a robust correlation between two phenotypes (e.g., alcohol consumption and alcohol withdrawal).Such a robust corre l a t i o n , which is re p resented by a corre l a t i o n coefficient r = 0.5-0.6,would mean that a p p roximately 25 percent of the va r iance observed in alcohol consumpt i o n resulted from variance in the other phenotype (i.e., withdrawal).
Studies involving inbred strains theref o re re q u i re a re l a t i ve l y large number of strains to detect moderate genetic correl a t i o n s .The analysis of numerous strains d e c reases the likelihood that differe n c e s b e t ween strains are caused by random fixation of alleles and increases the likelihood that the results also apply to other strains or organisms (i.e., can be g e n e r a li zed)-an important issue when one hopes to apply the re s u l t s to humans.Although such an analysis of numero u s strains is labor-and cost-intensive, it is the only way to identify correlated traits and calculate a corresponding corre l a t i o n coefficient.
The potential negative consequences of inbreeding can affect not only inbre d strains but also selected lines.Although s e l e c t i ve breeding usually specifically a voids mating brothers and sisters, i n b reeding still occurs, because the population used for creating a selected line (e.g., those animals showing the highest free-choice alcohol consumption) often is re l a t i vely small.In general, the

Some animals f rom high-pre f e r r i n g lines that have neve r been exposed to a l c o h o l s h ow lower l e vels of the bra i n chemical s e ro t o n i n .
smaller the population is in which s e l e c t i ve breeding is performed and the longer selective breeding is carried out, the greater is the level of inbre e d i n g ( Falconer and Mackay 1996).This i n b reeding can result in the generation of random differences (i.e., genetic drift) b e t ween two selected lines.For example, if some of the animals used for generating a high alcohol-consuming selected line by chance have a somewhat lighter coat color than the animals in the original population, the inbreeding inherent in generating the selected line may result in a population with a lighter coat color, even though coat color is u n related to alcohol consumption.The emergence of such differences in traits u n related to the phenotype of intere s t can greatly complicate the identification of genes that help determine the phenotype of interest (for a more mathematical presentation of this issue, see Belknap et al. 1997).Thus, to maximize the strength of s e l e c t i ve breeding-that is, to cause systematic differences related to the selection phenotype to emerge and to m i n i m i ze differences unrelated to the selection phenotype-experiments i n volving selected lines should maintain as large a population of bre e d i n g animals as is economically feasible.In addition, the generation of re p l i c a t e selected lines that are bred from differe n t animals to exhibit the same phenotype as the original lines can help re s e a rc h e r s i n t e r p ret differences between selected lines.A correlation that occurs in both the original selected lines and the re p l icate lines can be considered highly re l iable evidence that a true genetic correlation exists rather than a genetic drift ( Crabbe et al. 1990).

Qu a t i t a t i ve Trait Loci.
A n o t h e r a p p roach to examining the pleiotro p i c effects of alleles and dissecting genetic c o r relations in a particular population i n vo l ves the identification of quantitat i ve trait loci (QTLs).A QTL is a small section on the cell's genetic material (i.e., the DNA) that helps shape a quan-t i t a t i ve trait-in other words, a phenotype (e.g., alcohol consumption) that is determined by more than one gene, each of which exists in several alleles.Us i n g molecular genetic techniques, re s e a rc h e rs can locate such QTLs on the DNA and calculate the magnitude of their contribution to the phenotype under i n vestigation (for more information on QTLs, see the article in this issue by Grisel,.
Genetic correlation studies using selected lines and inbred strains to ident i f y the genes contributing to a cert a i n phenotype differ in several aspects fro m the QTL approach.First, QTL studies i n vestigate the influence of a single gene on the phenotype, whereas genetic correlation studies traditionally have been used to detect the influence of a gro u p of anonymous genes that act in concert and which may affect several phenotypes.In other words, selected lines and inbred strains a l l ow re s e a rchers to examine the entirety of a genetic corre l at i o n and do not attempt to dissect the genetic variance into pieces that are each influenced by one specific gene.This approach improves the statistical p owe r, and thus the re p l i c a b i l i t y, of t h e analysis, because it does not try to bre a k up genetic sources of variance into discre t e units, which can be difficult to detect re l ia b l y.The benefits of the genetic correlation approach are part i c ul a r l y g reat when analyzing phenotypes for which genetic factors play only a moderate role, such as alcohol consumption.For these phenotypes, each QTL may account only for a small port i o n of the variance in the phenotype, often less than 10 perc e n t .
C o n ve r s e l y, QTL studies may offer some advantages over genetic corre l a t i o n studies.For example, the failure to detect a genetic correlation using selected lines and inbred strains does not mean that such a correlation does not exist.In s t e a d , the correlation may be highly complex and invo l ve several genes having such d i verse (and even opposite) effects that they mask a genetic correlation when they act in concert.In such cases, the QTL approach may enable re s e a rc h e r s to find individual alleles that pro d u c e such opposite pleiotropic effects.In practice, howe ve r, scientists rarely take the trouble to look for QTLs underlying genetic correlations without having initially observed such correlations in animal models.In fact, both selected lines (e.g., Bice et al. 1998) and inbred strains f requently provide a starting point for QTL analyses, because these analyses are easiest when the DNA to be examined is derived from animals with two highly d i vergent phenotypes.

Transgenic and Knockout An i m a l s .
Two other appro a c h e s to examining the function of specific genes and their effects on alcohol-related traits in animal models invo l ve transgenic and knockout mice.In transgenic mice, the gene of i n t e rest is altered (i.e., mutated) in a test tube and then introduced into the mice, enabling re s e a rchers to study the effects of that gene alteration.In knockout mice, re s e a rchers inactivate the gene of i n t e rest, allowing them to draw conclusions on the function of that gene by determining the consequences of its absence.(For more information on these animal models, see the article in this issue by Bowers,.Like the QTL approach, these animal models attempt to analyze complex, alcohol-re l a t e d phenotypes by investigating the functions of individual genes.
In contrast to selected lines and i n b red strains, which take advantage of the genetic variability that occurs naturally in a population, transgenic mice i n vo l ve the generation of specific m u t ations in the gene of interest to cre a t e n ew variant alleles.Such targeted mutations are tremendously useful for a trait in which a known mutation exists in humans.This mutation can then be Selected lines and inbred strains a l l ow re s e a rchers to examine the e n t i rety of a genetic c o r re l a t i o n .
re p roduced in transgenic mice in ord e r to study its effects in more detail.In the vast majority of disorders (including alcoholism), howe ve r, re s e a rchers do not know the allelic va r i a t i o n that leads to d i f f e rences in human behavior.Theref o re, when attempting to model normal variations in humans that lead to d i f f e rences in alcohol-related behaviors, scientists may prefer to exploit the normal variation present in rodent populations rather than to create artificial genes by generating new mutants.
Fu rt h e r m o re, in contrast with normally occurring alleles found in selected lines and inbred strains, transgenic models using "a rt i f i c i a l" alleles may result in phenotypes that are influenced by specific (and often unanticipated) deve l o pmental and genetic parameters (for a discussion of this issue with respect to pain re s e a rch, see Mogil and Gr i s e l 1998).For example, re s e a rchers may experience difficulties in re p l i c a t i n g e ve n experiments in which the mutations had seemingly large effects, because small changes may have occurred in the DNA near the site of the mutation betwe e n the original and the replicate experiments (Phillips et al. 1999).There f o re , although transgenic models are undoubte d ly useful when identifying genes i n vo l ved in physiological functions, re s e a rchers should not overlook the i m p o rtance of normal variation in alleles that is re p resented by selected lines and inbred strains when attempting to genetically define important alcoholrelated phenotypes.

Summary
Selected lines and inbred strains, both of which rely on the normal genetic variability present within animal populations, continue to be useful tools in understanding the relationship betwe e n genes and alcohol-related traits.Mu c h w o rk remains to be done, howe ve r, in o rder to understand how genetic differences lead to behavioral differences in alcohol response.Genetically defined animal models, such as selected lines and i n b red strains, will continue to form the basis for this work.s